Premium
Identification of critical residues for transport activity of A cr3p, the S accharomyces cerevisiae A s( III )/ H + antiporter
Author(s) -
Markowska Katarzyna,
MaciaszczykDziubinska Ewa,
Migocka Magdalena,
Wawrzycka Donata,
Wysocki Robert
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13113
Subject(s) - antiporter , biology , transmembrane protein , transmembrane domain , biochemistry , transport protein , transporter , saccharomyces cerevisiae , yeast , membrane , receptor , gene
Summary A cr3p is an A s( III )/ H + antiporter from S accharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that C ys151 located in the middle of the fourth transmembrane segment ( TM 4) is critical for antiport activity, suggesting that A s( III ) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in A s( III )/ H + exchange, we performed a systematic alanine‐replacement analysis of charged/polar and aromatic residues that are conserved in the A cr3 family and located in putative transmembrane segments. Nine residues ( A sn117, T rp130, A rg150, T rp158, A sn176, A rg230, T yr290, P he345, A sn351) were found to be critical for proper folding and trafficking of A cr3p to the plasma membrane. In addition, we found that replacement of highly conserved P he266 ( TM 7), P he352 ( TM 9), G lu353 ( TM 9) and G lu380 ( TM 10) with Ala abolished transport activity of A cr3p, while mutation of S er349 ( TM 9) to A la significantly reduced the A s( III )/ H + exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of G lu353 and G lu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM 9 and TM 10 are crucial for antiport activity. We also discuss a hypothetical model of the A cr3p transport mechanism.